This project is focused on deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the diphtheria toxin T-domain (DTT), which is considered to be a paradigm for cell entry of other toxins (e.g., tetanus and botulinum) and has a potential for targeted delivery of anti-cancer therapies. The pH-triggered insertion of DTT will also reveal general physicochemical principles underlying membrane protein assembly and signalyng on membrane interfaces. This first competing renewal of the project will capitalize on our progress in identifying key intermediate states along the insertion pathway, in establishing the concept of conformational switching for DTT action and in developing new methodologies for structural, kinetic and thermodynamic characterization of membrane protein refolding/insertion. The innovation of this proposal resides in the unique way that molecular dynamics (MD) simulations and sophisticated spectroscopic experiments will be brought together in order to understand molecular mechanisms which will bring clarity to a complex field. MD simulations will be used for (a) building atomic models consistent with low resolution spectroscopic data, and (b) guiding the experimental design to further verify them. Site-specific labeling of single-cysteine mutants and a battery of spectroscopic approaches (including FCS, fluorescence lifetime quenching, FRET, stopped-flow kinetic measurements) will be utilized to test the interface-directed refolding/insertion hypothesis, which assigns a special role to the bilayer interfacial region in modulating transmembrane insertion by assisting the formation of key intermediate states, shifting the balance of electrostatic and hydrophobic interactions and altering protonation properties of titratable residues. The nature of the conformational switching resulting in refolding, insertion and translocation transitions of DTT will be established through mutagenesis of His, Asp and Glu residues, guided by Thermodynamic Integration calculations. Various DTT mutants will be used to ascertain whether protonation of histidines assists in the unfolding of the protein in solution and promotes formation of a previously identified insertion-competent intermediate on the membrane interface, through electrostatic interactions with anionic lipids, while protonation of acidic residues enables transmembrane insertion. To gain insights into the pH-triggered membrane action of DTT, thus establishing the general physicochemical principles of membrane-protein interactions, we will pursue the following goals: (1) determine molecular details of the structural organization of key intermediate and final inserted states; (2) determine the free energy profile of transitions along the insertion pathway and determine how the properties of the bilayer modulate structural, thermodynamic and kinetic parameters of the DTT insertion; and (3) identify key residues responsible for pH-triggered functional conformational switching.